1. Field of the Invention (Technical Field)
The present invention relates to dual microsensors for the simultaneous amperometric monitoring of glucose and insulin, and to oxygen-rich composite enzyme electrodes and their use in a range of substrate concentrations and environments severely depleted in oxygen.
2. Background Art
Diabetes mellitus is a chronic metabolic disorder that results from a total or partial deficiency of insulin. In order to manage care, including providing enhanced diabetes control, there is a recognized need for glucose monitoring, including the simultaneous detection of glucose and other analytes, including most particularly insulin. Insulin sensing is of significant importance for clinical diagnosis, because it serves as a predictor of diabetes, insulinoma and trauma. By simultaneously measuring both glucose and insulin, an insulin/glucose ratio can readily be determined, thereby providing a single point assay for diagnosis of insulinoma and the management of diabetes.
It is known to utilize a ruthenium-oxide (RuOx)-type catalytic film sensor for insulin detection at physiological pH. Gorski, W., Aspinwall, C., Lakey, J. R. T., Kennedy, R. T., J. Electroanal. Chem. 1997, 425, 191. RuOx sensors have been used, for example, to monitor insulin secretion from pancreatic β cells. However, this device cannot be used to detect glucose or other substances.
Oxidase-catalyzed enzymatic reactions play a major role in the development of enzyme-based biosensors (Clark, Jr., L. C., Sachs, G. Ann NY Acad. Sci. 1962, 148,133; Schultz, J. Sci. Amer. 1991(8), 64). The large number of commercially available oxidases opens up the prospects for the detection of important substrates (such as glucose, lactate, or cholesterol) in relevant clinical, food or biotechnological matrices.
The following reaction: can be monitored amperometrically: As the immobilized enzyme relies on the use of oxygen as the co-substrate, the operation of these enzyme electrodes suffers from problems due to restricted solubility of oxygen and variations in the oxygen level (Zhang, Y., Wilson, G. S. Anal. Chim. Acta 1993, 281,513). For example, implantable glucose sensors often suffer from low oxygen availability in subcutaneous tissue.
Common routes for minimizing the strong oxygen dependence include the replacement of oxygen with a non-physiological electron acceptor (Cass, A. E., Davis, G., Francis, G., et al., Anal. Chem. 1984 56, 667), or the use of a proper membrane coverage that improves the surface availability of oxygen (relative to the substrate) (Gough, D., Lucisano, J., Tse, P. Anal. Chem. 1985, 57, 2353; Fischer, V., Hidde, A., Herman, S., von Woedtke, T., Rebrin, K., Abel, P. Biomed. Biochim. Acta 1989,48,965).
Others in the field have approached the problem of a limited oxygen environment in various ways: Clark (U.S. Pat. No. 4,721,677 (1988)) relies upon an oxygen chamber with a membrane to hold oxygen extracted from the tissues; Zawodzinski et al. (U.S. Pat. No. 5,227,042 (1993)) utilizes a back supply (bubbling) of oxygen through a porous electrode; and Rishpon et al. (U.S. Pat. No. 5,082,550 (1992)) casts a perfluorosulfonic acid isomer as a film directly on the conductor surface of the electrode to supply oxygen. These approaches sometimes result in a rather bulky apparatus and cumbersome operation, and are not compatible with miniaturization. In addition, they do not address the additional problem of electroactive interferences that can strongly affect sensor output.
Others in the field who address the interference problem (but ignore the oxygen limitation problem) frequently use a platinized carbon to create a paste for the enzyme (Bennetto et al., U.S. Pat. No. 4,970,145 (1990); Maley et al., U.S. Pat. No. 5,616,222 (1997)). These methods decrease interference somewhat, but still function at higher potentials.
Carbon paste electrodes (CPEs), consisting of a mixture of graphite powder and an organic pasting liquid (commonly mineral oil), represent an attractive approach for the preparation of reagentless biosensors (Wang, J., Un, M. S. Anal. Chem. 1988, 60, 1545; Gorton, L. Electroanalysis 1995, 7,23). The pasting liquid serves not only for filling the crevices between the graphite particles, but results in an electrode that is fundamentally different from those (e.g., Pt, Au) commonly used for amperometric transduction. Common pasting liquids include mineral oil, parrafin oil, silicone grease, and bromonaphthalene.
U.S. Pat. No. 5,922,183 (1999), to Rauh, discloses a thin film matrix for detecting substances, including glucose, utilizing an enzyme biosensor, such as a glucose oxidase, and a hydrous metal oxide as a catalyst or “cofactor” in enzyme reactions. However, this approach does not provide for simultaneous detection of glucose and insulin utilizing catalytic detection of insulin.
This invention provides for a combined needle-type probe, integrating an amperometric glucose biosensor with an electrocatalytic insulin microsensor. Thus in the same needle body an insulin-sensitive RuOx-modified carbon-paste microelectrode is integrated with a metalized, such as rhodinized, carbon amperometric glucose b-biosensor. Despite substantial analyte concentration differences, with mM concentrations of glucose and nM concentrations of insulin, and the use of different transduction principles, the microsensors of the invention respond independently and rapidly to the corresponding target analytes, with no apparent cross reactivity.
This invention further successfully addresses problems encountered in many in vivo and in vitro monitoring electrodes, including oxygen dependence of oxidase enzyme electrodes and redox interferences, through the use of carbon paste biosensors with oxygen-yielding (e.g. fluorochemical) pasting liquids supplied in a reservoir within the needle-type sensor. Due to the high oxygen solubility in perfluorocarbons, such pasting liquids provide an internal supply of oxygen, and efficient operation of first-generation oxidase electrodes under oxygen-deficient conditions. The result is a composite (binder serves as the oxygen reservoir) bioelectrode that provides real-time in vivo or ex vivo, accurate monitoring of specific chemical environments within the body.